1. Field
This disclosure relates to specimen analysis through the use of ellipsometry. More particularly, the present disclosure describes improvements in ellipsometric performance during specimen analysis by reducing the impact of birefringence.
2. Description of Related Art
There are numerous categories of procedures under the general heading of bioassay. Here is a list that is at least exemplary if not exhaustive of the type of bioassays in which the technology known as imaging ellipsometry or sometimes evanescent field imaging is or can be applied:
1. Label free DNA hybridization assay. Microarray of single strand DNA can be placed on the surface of the substrate. The binding of the corresponding strand from a sample under study can be detected. The implementation is often used for DNA sequencing and called a DNA chip.
2. Label free protein assay. Microarray of proteins can be placed on the surface of the substrate. The binding of the corresponding molecular target can be detected. If the binding molecule is disease related, often peptides, protein produced by the disease, by the host, the protein assay can be used for diagnostic purposes.
3. Small molecule discovery assay. A panel of possible biomarker drug candidates can be placed on the substrate. A sample containing the target, often a protein that would trigger a cascade reaction, would be applied and the selective binding can be observed.
4. Label free micro-organism capturing assay. Molecules, for example, targeting specific micro-organism can be placed on the surface of the substrate. When the solution containing the specific organism, such as cells, parasites and bacteria, is applied, the organism captured at the surface can be detected. A micro-organism capturing assay can be used for capturing, diagnostic or environmental purposes. The molecule placed on the substrate can be protein, lipid, sugar, bio-inert molecules such as PEG, or a composition of these molecules.
5. Cell-based assay. Cells can be placed on the surface of the substrate. Stimulation is introduced to the cells, and the reaction is measured through the reaction footprint of the cells. The dosage and efficacy of the stimulation can be quantified through the reaction of the cells. The typical cell-based assay includes proliferation assay, wound healing assay, toxicity assay. Also the indirect target of the stimulation can be studied through performing the cell-based assay. For example, genetically engineered cells which over or under express a targeted protein can have medicated reaction under the reaction of the stimuli.
U.S. Pat. No. 6,859,280 discloses an imaging ellipsometry system which employs a transparent substrate and directs polarized light at the upper surface of the substrate in a manner to produce total internal reflection (TIR) and an evanescent field at the upper surface of the substrate. The system disclosed in U.S. Pat. No. 6,859,280 is designed to sense chemical reactions occurring in an evanescent field at the upper surface of the substrate by sensing intensity changes at locations of the surface which correspond to pixel locations in a two-dimensional sensor such as a charge coupled device (CCD) to which reflected light is directed. The chemical reactions occur by immobilizing an array of receptors on the upper surface of the substrate and sensing changes in polarization of the reflected light due to binding events between analytes (ligands) in a sample introduced to the surface and receptors in the array. Alternatively, a drug is introduced to cells cultured at the upper surface of a transparent substrate for analysis as, for example, by a dose response to carbachol, a cholinergic receptor agonist, resulting in changes to the cells' footprint on the substrate surface sensed as localized intensity changes in the reflected light. Cells are naturally spatially distributed. By producing an image, it allows selective analysis of the changes in the polarization state in the cross-section of the reflected beam which are indicative of the substances in the specimen in the location corresponding to a position in the detector. Therefore, the system may be configured to produce an image which represents the change in polarization state within each of the discrete specimen spots or within the footprint of cultured cells. That change can then be output as an image of physical measurement of physical properties such as density or height. The image refers to a set of values correlated to different spatial positions which can be displayed or compared against each other to contrast differences in physical properties at these positions.
Multiwell disposables used to contain specimens for analysis are commonly available. For ellipsometric systems where polarized light is employed in the imaging system, the disposable (slide or well) should be birefringence-free to avoid significant reduction of sensitivity in the detection (imaging) of binding events. Birefringence free disposables are usually made from glass and are too expensive to be used in large segments of the market for ellipsometric systems. Plastic disposables, on the other hand, are sufficiently inexpensive to be useful in virtually every segment of the market. But plastics, although promising, presently exhibit birefringence which reduces the sensitivity of system output signals and thus the usefulness of the systems.
As indicated above, most existing ellipsometry implementations require a high performance optical material as the substrate which does not alter the relative phase of the two polarizations outside the binding surface. The high performance requirement is an obstacle in reducing the cost of performing ellipsometry and is not always achievable in common optical material. Birefringence in material, which is a phenomenon where the speed of light depends on its direction, can contribute to the polarization phase measured and skew the measured result on binding. That is, birefringence in the material on which a specimen is distributed will skew the polarization phase changes of the light transmitted to the sensor. Hence, the measured polarization may not accurately reflect the polarization change arising from biochemical interactions.
Therefore, there exists a need to improve the collection and processing of birefringence-impacted ellipsometry data to allow for lower cost optical material to be used and/or to allow for faster processing of the data.